This grant application seeks to better understand the role for microRNAs in the molecular mechanisms by which cocaine remodels the striatum and thereby drives the emergence of addiction-relevant behavioral abnormalities. A major accomplishment during the previous funding period was showing that disruption of the microRNA biogenic machinery in striatum profoundly decreases cocaine intake in genetically modified mice. This finding supports a key role for striatal microRNAs in regulating the reinforcing properties of cocaine. Subsequently, we established that two closely related microRNAs, miR-212 and miR-132, are induced in the striatum of rats demonstrating compulsive-like cocaine-taking behaviors. miR-212 exerts an inhibitory influence on cocaine-taking behavior through a mechanism involving enhanced striatal CREB signaling and diminished MeCP2/BDNF signaling. By contrast, our recent data suggests that miR-132 may enhance the motivational properties of cocaine through, as yet, unclear mechanisms. We hypothesize that the balance between miR-212 and miR-132 signaling influences resilience vs. vulnerability to addiction. In this competitive renewal, we will use cutting edge molecular, cellular and behavioral approaches to define the precise mechanisms by which miR-212 and miR-132 control drug intake. The research plan builds logically and innovatively on the progress made during the last funding period. In Specific Aim I, the cellular mechanisms by which miR-212 and miR-132 act in striatum will be investigated. To accomplish this aim we have successfully constructed two new lines of transgenic mice: those with ?floxed? alleles for the miR-212 or the miR-132 gene. Using these mice, we will conditionally delete miR-212 or miR-132 in D1 receptor-expressing medium spiny neurons (D1-MSNs) or in D2-MSNs, the two major cell populations that together represent ~95% of total neurons in striatum. The effects of these cell-specific lesions on cocaine self-administration, and other addiction-relevant behaviors, will be investigated. In Specific Aim II, the molecular mechanisms by which miR-212 and miR-132 control cocaine intake will be investigated. We will use High-Throughput Sequencing of RNA isolated by CrossLinking ImmunoPrecipitation (HITS-CLIP) to identify genes targeted by miR-212 and miR-132 in D1-MSNs and D2- MSNs. We will verify that identified genes are direct targets for miR-212 and/or miR-132 using 3?UTR luciferase reporter assays, RNA expression analysis and protein immunoblotting. Under Specific Aim III, we will use in vivo CRISPR technology to investigate the contribution of the most promising gene targets whose expression is controlled by miR-212 and/or miR-132 in regulating the motivational properties of cocaine. These studies promise to yield significant new insights into molecular and cellular mechanisms of cocaine addiction.